Bone is a unique type of tissue composed of an inorganic mineral phase and cellular and extracellular matrix phases. Bone is a vital organ that undergoes modeling and remodeling wherein old bone is lost (resorption) and new bone is formed (replacement).
In children and young adults, bone remodeling results in the growth and increase in density of the skeleton. In adults, remodeling normally results in no net change in skeletal size since bone replacement matches bone resorption. Osteoporosis and related diseases ensue when bone resorption exceeds bone replacement. Bone restoration or replacement is a viable consideration in indications including osteopenia, osteoporosis, bone tumors, spinal fusion and fractures.
Each year, more than 6.3 million people in the U.S. experience bone fractures, of which almost 1 million require hospitalization. Natural healing of bone with mechanical fixation can, in most cases, adequately mend minor fractures over time. However, in approximately 10% of all fractures, the defect is too large for the body's natural healing response, and delayed unions or non-unions develop at the fracture site (Bancroft and Mikos, 2001; Rozen et al., 2007). In these cases, supplementary bone material may be required to fill in the defect and restore structure and function.
Current Therapies for Treatment of Severe Bone Defects
The standard treatment for healing severe bone defects is transplantation of autologous bone tissue (autografts) (reviewed in Mistry and Mikos, 2005). However, this process has several drawbacks including: limited body sites from which bone may be harvested without loss of function; autografts are less effective in irregularly shaped defects; and the procedure may be associated with complications such as infection, pain and nerve injury. Allografts derived from cadavers are another commonly used bone graft material. However, un-processed grafts carry a risk of disease transmission and immune rejection, while demineralized bone matrix is poor in bone growth inducing factors. Xenografts are also a poor option due to the danger of disease transmission or rejection.
Metal implants may be permanently placed in bone to fill a defect however corrosion, infection and poor implant-tissue interface create many problems.
Ceramics may also be used in the treatment of bone injuries. While offering excellent biocompatibility, they are often brittle and degrade too slowly thereby inhibiting natural bone re-growth.
Another potential treatment is distraction osteogenesis, which entails the lengthening of limbs across a defect through temporary external fixation devices.
As outlined above, the most advanced treatments are limited in effectiveness and are often associated with complications. Thus, there is a significant need for an alternative strategy for the treatment of severe bone loss or fracture.
Cell-Based Therapies
An emerging approach to damage repair is tissue engineering, which involves treatment with one or more of the following elements: cells, signaling molecules and scaffolds. Thus, by employing the body's natural healing response, a bone defect may be replaced by natural bone tissue in the absence of an exogenous permanent implant (Bancroft and Mikos, 2001; Rozen et al., 2007).
Cell-based strategies for bone tissue engineering involve, inter alia, the transplantation of osteogenic cells. Cell-based therapies may include fresh bone marrow, as well as mesenchymal stem cells (MSCs) expanded in culture, or differentiated osteoblasts. Autologous bone-marrow injected into patients' un-connected tibial fractures with fixation demonstrated efficacy equal to that of autografts (Connolly et al., 1991).
A key factor for the success of bone marrow in healing non-union fractures is the presence of MSCs. The limited quantity of MSCs in marrow has led to the development of methods to isolate progenitor cells from bone marrow and expand them in-vitro, prior to transplantation.
Richards et al., (1999) demonstrated the osteogenic capabilities of cultured MSCs in a collagen gel carrier injected into distraction gaps of rats. Kadiyala et al., (1997) loaded MSCs onto ceramic cylinders and implanted them into critical-sized defects in rat femora. In U.S. Pat. Nos. 6,541,024 and 6,863,900 to Kadiyala et al. regeneration and augmentation of bone repair following administration of MSCs was disclosed.
Isolated MSCs in culture can be selectively differentiated into osteoblasts with media supplements (Bruder et al., 1997; Pittenger et al., 1999). These osteogenic cells, when combined with a biomaterial carrier, can begin bone reconstruction immediately upon delivery to the injury site. For example, when rat marrow stromal cells cultured on porous hydroxyapatite scaffolds with osteogenic supplements were implanted subcutaneously in rats, rapid bone formation was observed (Yoshikawa et al., 1996).
U.S. Pat. No. 5,811,094 (Caplan et al.) provides a method for isolating, purifying, culturing and expanding human mesenchymal stem cells (MSCs) for the purpose of repairing connective tissue defects (including bone and cartilage repair). U.S. Pat. No. 6,355,239 (Bruder et al.) provides methods and preparations for promoting connective tissue growth, including bone, by transplanting allogeneic, mesenchymal stem cells.
U.S. Pat. No. 7,029,666 (Bruder et al.) demonstrates use of non-autologous MSCs for treatment and regeneration of connective tissue and enhancement of bone marrow engraftment.
U.S. Pat. No. 5,972,703 (Long et al.) discloses a process for preparing an enriched population of bone precursor cells (expressing osteocalcin or osteonectin) obtained from bone marrow for promoting bone fracture repair.
Endothelial Progenitor Cells
Endothelial progenitor cells (EPCs) have been identified in adult bone marrow as well as in peripheral blood and human umbilical cord blood, and have been shown to maintain their potency to proliferate and to differentiate into mature endothelial cells (Ashara et al., 1997; Murohara et al., 2000). Vasculogenesis, the development of new blood vessels during embryogenesis begins with the formation of blood islands comprising endothelial progenitor cells (EPCs) and hematopoietic stem cells (Risau, 1997; Risau, 1995; Risau et al., 1988; Flamme et al., 1992; Hatzopoulos et al., 1998; Doyle et al., 2006; Ribatti, 2007).
EPCs have been shown to participate in postnatal neovascularization (Takahashi et al., 1999; Isner and Asahara, 1999). Furthermore, EPCs were found to participate in angiogenesis, vascular repair and vasculoprotection (Humpert et al., 2005; Doyle et al., 2006).
Recent studies have shown that EPCs significantly participate in constructing endothelium of new vessels in situations of tissue regeneration such as burns, bypass coronary artery grafting, and acute myocardial infarction. In these instances, bone marrow-derived EPCs are recruited to the blood circulation and home to injured and regenerating tissues for their participation in the buildup of new blood vessels. For example, addition of a purified and ex-vivo expanded population of these cells to nude mice with hind limb ischemia improved blood flow recovery and reduced limb loss (Kalka et al., 2000). Moreover, growth factors and ischemic conditions augment the number of circulating EPC (Takahashi et al., 1999).
EPCs can be identified by tube formation in Matrigel™ (Bellik et al., 2005), acetylated low-density lipoprotein (Ac-LDL) incorporation or expression of characteristic cell markers including Tie-2, CD34+ and von Willebrand factor (vWf) (Neumuller et al., 2006).
Early studies in a distraction osteogenesis model in sheep described the appearance of cellular colonies of vascular nature (immunopositive for Tie-2 and factor VIII-related antigen), the origin of which was not clear (Rachmiel et al., 2002; Lewinson et al., 2001(a, b)).
Cetrulo et al., (2005) demonstrated that distraction osteogenesis in a rat mandible model results in the generation of an ischemic region, to which concomitantly injected human EPCs were shown to home.
U.S. Pat. No. 6,878,371 to Ueno et al. provides methods of forming new blood vessels in diseased or damaged tissue, specifically cardiac muscle comprising transplanting locally autologous bone marrow mononuclear cells.
Vascular injury was shown to promote an increase in circulating endothelial cells and EPCs (Hunting et al., 2005). In another study, bone marrow derived circulating EPCs were used to enhance angiogenesis following tissue ischemia (Werner et al. 2006).
U.S. Pat. No. 6,720,340 (Cooke et al.) discloses recruitment of bone marrow derived EPCs and hematopoietic stem cells to the site of deficiency or injury, by administration of nicotine or nicotine receptor agonists.
Nowhere in the background art is it taught or suggested that local transplantation of autologous endothelial progenitor cells augments bone regeneration and may therefore be used as a therapeutic strategy for critical-gap bone fracture repair. There remains an unmet need in the medical community for an effective treatment for non-union and delayed union fractures.